BACKGROUND: Termitomyces heimii is a basidiomycete fungus that has a symbiotic relationship with termites, and it is an edible mushroom with a unique flavour and texture. T. heimii is also one of the most difficult mushrooms to cultivate throughout the world. Little is known about the growth and development of these mushrooms, and the available information is insufficient or poor. The purpose of this study was to provide a base of knowledge regarding the biological processes involved in the development of T. heimii. The proteomic method of 2 dimensional difference gel electrophoresis 2D-DIGE was used to determine and examine the protein profiles of each developmental stage (mycelium, primordium and fruiting body). Total proteins were extracted by TCA-acetone precipitation. RESULTS: A total of 271 protein spots were detected by electrophoresis covering pH 3 - 10 and 10 - 250 kDa. Selected protein spots were subjected to mass spectrometric analyses with matrix-assisted laser desorption/ionisation (MALDI TOF/TOF). Nineteen protein spots were identified based on peptide mass fingerprinting by matching peptide fragments to the NCBI non-redundant database using MASCOT software. The 19 protein spots were categorised into four major groups through KEGG pathway analysis, as follows: carbohydrate metabolism, energy metabolism, amino acid metabolism and response to environmental stress. CONCLUSIONS: The results from our study show that there is a clear correlation between the changes in protein expression that occur during different developmental stages. Enzymes related to cell wall synthesis were most highly expressed during fruiting body formation compared to the mycelium and primordial stages. Moreover, enzymes involved in cell wall component degradation were up-regulated in the earlier stages of mushroom development.